背景:干细胞移植为慢性肾脏病的治疗提供了新的视角,特异性标记干细胞并体内示踪是这一领域研究的基础,目前尚无一种公认的适用于所有细胞的标记方法。目的:观察DAPI及绿色荧光蛋白标记细胞在阿霉素肾病大鼠体内的定位及分化,探讨便捷的大鼠胚胎后肾间充质细胞的体外标记方法。设计、时间及地点:分组对比观察,于2007-04/12在中南大学湘雅二医院完成。材料:清洁级雌性Sprague-Dawley(SD)大鼠60只,体质量180～220g,用于制备阿霉素肾病大鼠模型。方法:分别将DAPI、DAPI体外标记后肾间充质细胞、绿色荧光蛋白体外标记后肾间充质细胞,通过尾静脉注入阿霉素肾病大鼠体内;于细胞移植后1,3,5周行肾组织冰冻切片,观察DAPI及绿色荧光蛋白荧光分布,并ABC免疫酶染色法检测肾组织中绿色荧光蛋白表达。主要观察指标:比较DAPI及绿色荧光蛋白两种不同方法标记后肾间充质细胞在阿霉素慢性肾病大鼠肾内的定植情况,以及绿色荧光蛋白标记细胞不同时间点移植在肾内的定植改变。结果:①DAPI标记细胞及单纯DAPI尾静脉注射后1周,均可在肾小管上皮细胞中观察到DAPI阳性细胞,至移植后5周仍可观察到,但随时间延长荧光有减弱趋势。②绿色荧光蛋白标记细胞移植后1周,可在肾小管上皮细胞中观察到绿色荧光蛋白阳性细胞,至移植后5周仍可观察到,荧光无明显减弱。③ABC免疫酶染色法检测,显示绿色荧光蛋白阳性细胞主要表达于近端肾小管中,肾小球系膜区亦可观察到少量表达;绿色荧光蛋白阳性产物主要表达于胞浆。绿色荧光蛋白表达半定量评分显示,标记细胞的早期应用阳性率大于晚期应用者,且随观察时间延长,阳性率有升高趋势。结论:后肾间充质细胞的脂质体感染法绿色荧光蛋白标记是一便捷、有效的体外标记、示踪方法,适于移植细胞的短期内示踪、观察。 BACKGROUND: Stem cell transplantation provides a new perspective for the treatment of chronic kidney disease. Specific labeling of stem cells and in vivo tracing is the basis of research in this field. Currently, there is no accepted method for labeling all cells. OBJECTIVE: To observe the localization and differentiation of DAPI and GFP-labeled cells in adriamycin-induced nephropathy rats and to explore the method of in vitro labeling of rat embryo-derived renal mesenchymal cells. DESIGN, TIME AND SETTING: The group comparison observation was performed at Xiangya Second Hospital, Central South University from April to December in 2007. MATERIALS: Sixty female Sprague-Dawley (SD) rats of clean grade with a body weight of 180-220 g were used to prepare adriamycin-induced nephropathy rat model. Methods: DAPI and DAPI were used to label in vitro renal mesenchymal cells labeled with mesenchymal cells and green fluorescent protein in vitro. Adrenocortical nephropathy rats were injected via the tail vein respectively. At 1, 3, 5 weeks after cell transplantation Frozen sections of renal tissue were observed. The fluorescence distributions of DAPI and green fluorescent protein were observed. The expression of green fluorescent protein (GFP) in renal tissues was detected by ABC immunohistochemical staining. MAIN OUTCOME MEASURES: Colonies of renal mesenchymal cells labeled with DAPI and GFP were compared in the kidneys of adriamycin-induced chronic kidney disease rats and colonization of the kidney with transfection of green fluorescent protein-labeled cells at different time points change. RESULTS: ① DAPI-positive cells were observed in renal tubular epithelial cells one week after DAPI-labeled cells and DAPI-only intravenous injection. However, DAPI-positive cells could still be observed 5 weeks after transplantation. However, the fluorescence decreased with time. ② GFP labeling One week after cell transplantation, green fluorescent protein positive cells could be observed in renal tubular epithelial cells, and still can be observed 5 weeks after transplantation, the fluorescence was not significantly weakened. ③ ABC immunohistochemical staining showed that green fluorescent protein positive cells were mainly expressed in the proximal tubules, and glomerular mesangial area was also observed a small amount of expression; the positive products of green fluorescent protein were mainly expressed in the cytoplasm. Semi-quantitative green fluorescent protein expression score showed that the positive rate of labeled cells in early stage was higher than that in advanced stage, and the positive rate increased with the extension of observation time. CONCLUSION: Green fluorescent protein labeling of mouse hindgland mesenchymal cells by liposomes is a convenient and effective method for labeling and tracing in vitro. It is suitable for short term tracing and observation of transplanted cells.